Western Blot Tips and Tricks slides_Western...Western Blot Tips and Tricks Filling the Gap Between...
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Western Blot Tips and Tricks
Filling the Gap Between Art and Science
Instructions for Viewers
Webinar Series
July 30, 2014
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Brought to you by the Science/AAAS Custom Publishing Office
Sponsored by:
Participating Experts
Pier Giorgio Righetti, Ph.D.
Polytechnic University of Milan
Milan, Italy
Biji T. Kurien, Ph.D.
University of Oklahoma Health
Sciences Center
Oklahoma City, OK
Webinar Series
Nick Thomas, Ph.D.
GE Healthcare
Cardiff, Wales
Western Blot Tips and Tricks
Filling the Gap Between Art and Science
July 30, 2014
![Page 3: Western Blot Tips and Tricks slides_Western...Western Blot Tips and Tricks Filling the Gap Between Art and Science Instructions for Viewers Webinar Series July 30, 2014 Brought to](https://reader031.fdocuments.us/reader031/viewer/2022021811/5c955b0d09d3f29c7b8c906f/html5/thumbnails/3.jpg)
Pier Giorgio Righetti
Department of Chemistry, Materials and
Chemical Engineering “Giulio Natta”, Politecnico di Milano, Via Mancinelli 7, 20131
Milano, Italy
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• Most likely is the protein load • Typically as low as 1 ng, the detection limit of silver staining • At such low loads, blots can be poorly reproducible, due to:
Antigen losses already during sample homogenization and centrifugation of cell debris
Losses due to adsorption onto glass and plastic walls of sample containers
Losses due to sample adsorption by the polyacrylamide gel fibers in SDS-PAGE or IEF in IPGs or 2D mapping
Incomplete electrophoretic transfer to the blotting membrane • Due to all above accidents, it is no wonder that for trace antigens
Western blot analysis might be unsuccessful!
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• Subtraction approach: immuno-depletion of the 20 high-abundance proteins in
biological fluids (notably serum, urines, cerebrospinal fluid). The problems? • Capability of handling only minute sample volumes (max 100 µL) • Sample dilution after the depletion process • Therefore only very poor gain in visibility of trace proteins
• Enrichment approach: enabling capture of specific classes of proteins, e.g.
glycoproteins, phosphoproteins or an “ecumenic” approach permitting capture of all sample proteins by implementing a substantial reduction of the dynamic range. This approach is the “combinatorial peptide ligand library (CPLL) methodology”. Its advantages:
• Capability of handling very large sample volumes (up to 1 L) • Simultaneous reduction of high-abundance proteins (HAP) and high enrichment of
low-abundance proteins (LAP) • Increment of visibility for LAPs of up to 3 to 4 orders of magnitude!
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The hexapeptides are
bound to an organic
polymer,
poly(hydroxymethyl
acrylate) all throughout
the core of the pearls.
Some 64 million diverse
baits are present on the
beads, enough to
capture just about any
protein expressed in
any proteome!
Note that the
hexapeptides terminate
with a D-amino acid!
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Initial sample (large dynamic range)
Ligand library
Loading
a b c d
Adsorption
FT fraction
Wash away unbound sample
Recover bound protein
Collected sample (diminished dynamic range)
1
2
3
4
Ctrl.
FT
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• When complex samples are incubated with the beads, proteins bind to their specific ligands.
• High abundance proteins quickly saturate their specific ligands.
• High abundance proteins cease binding upon reaching their saturation point.
• Unbound proteins in excess, are removed during wash step.
• Medium and low abundance proteins continue to bind to their ligands.
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Reduction of dynamic range by CPLL Beads
• Simultaneous dilution of HAP and concentration of LAP;
• In a single and reproducible step;
• No species depletion is contemplated with this method;
• “Normalization” exclusively achievable using bead libraries of a very large diversity;
• A controlled sample-to-bead ratio allows a given reduction in dynamic range of the proteome.
Range of Detection
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Visibility of 2 SAA spots starts at 5-10 ng/μL concentration
Zoomed areas of control samples - untreated
0 ng/μL SAA
160 ng/μL SAA 80 ng/μL SAA 40 ng/μL SAA
10 ng/μL SAA 5 ng/μL SAA 1 ng/μL SAA
20 ng/μL SAA
8-16% Tris-HCl gels
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Visibility of 2 SAA spots starts at 1 ng/μL concentration (note that the OD of one of this spots is similar to the density of the 80 ng/μL in the untreated sample of the previous slide)!
Zoomed areas of eluted samples – treated with CPLLs
16% Tris-HCl gel
0 ng/μL SAA
160 ng/μL SAA 80 ng/μL SAA 40 ng/μL SAA
10 ng/μL SAA 5 ng/μL SAA 1 ng/μL SAA
20 ng/μL SAA
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Figure 3 - Fasoli et al.
3 pH 103 pH 10
1
2
3
4
5
250
150
50
37
25
20
15
10
75
kDa
100
3 pH 10
1
2
3
4
5
1L
2R
6
7
8
3 pH 10
10
10
Blot
100
75
50
37
2520
15
10
150
250
kDa
Fig. 2 – Fasoli et al.
New allergenic proteins:
• Vicilin
• globulin-2
• endo-chitinase
• thioredoxin
• trypsin inhibitor
Blotted proteins revealed with radio-iodinated antibodies (IgE) against sera of 3 different maize-allergic patients
Zea-mays B73
Lipid transfer protein (LTP)
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Control: only one antigen Pers a 1; After CPLL treatment: five more: profilin, a polygalacturonase, a thaumatin-like protein, a glucanase, and an isoflavone
reductase-like protein
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Known allergens in control, untreated sample: Mus a 1 (profilin), Mus a 2 (class I chitinase), Mus a 4 (thaumatin-like protein), and Mus a 5 (β-1,3-
glucanase).
New allergens after CPLL treatment: a pectinesterase and a superoxide dismutase
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Control: 374 unique gene products
CPLL Treated: 2855 unique proteins
Known allergens in control: no one reported so far;
New allergens after CPLL treatment: non-specific lipid transfer protein, superoxide dismutase, germin-like protein and profilin.
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Brought to you by the Science/AAAS Custom Publishing Office
Sponsored by:
Participating Experts
Pier Giorgio Righetti, Ph.D.
Polytechnic University of Milan
Milan, Italy
Biji T. Kurien, Ph.D.
University of Oklahoma Health
Sciences Center
Oklahoma City, OK
Webinar Series
Nick Thomas, Ph.D.
GE Healthcare
Cardiff, Wales
Western Blot Tips and Tricks
Filling the Gap Between Art and Science
July 30, 2014
![Page 19: Western Blot Tips and Tricks slides_Western...Western Blot Tips and Tricks Filling the Gap Between Art and Science Instructions for Viewers Webinar Series July 30, 2014 Brought to](https://reader031.fdocuments.us/reader031/viewer/2022021811/5c955b0d09d3f29c7b8c906f/html5/thumbnails/19.jpg)
Tips and challenges in
western blotting
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Western blotting
Transfer of protein patterns from
gel to microporous membranes
Electrophoretic-Towbin, Burnette
Burnette, to retain “geographic”
naming tradition initiated by Ed
Southern’s paper
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Advantages of transfer to
membrane
Wet membranes are pliable and
are easy to handle
This allows easy accessibility of
proteins immobilized on membrane
to different ligands
Only small amount of reagents
required for transfer analysis
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Advantages (cont’d)
Multiple replicas of a gel possible
Prolonged storage of transferred
patterns
Same protein transfer for multiple
successive analyses
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http://dna-protein.blogspot.com/2012/07/the-good-bad-
and-ugly.html
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Considerations for a
good western blot
Proper sample preparation
Choice of right lysis buffer
Proper gel preparation
Right electrophoresis and
transfer conditions
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Sample preparation
From cultured cells
Washed with PBS to remove
media
Remove PBS prior to cell lysis
Salt contamination causes
horizontal streaking in 2 DE
Westermeier R. Practical Proteomics, 2007;
Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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Eliminating viscosity from
DNA in crude extracts
Treat extracts with protease
free nuclease (Benzonase)
Shearing with sonication
Vigorous vortexing of heated
sample
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Protein solubilization:
Choice of Lysis buffers
Laemmli (cell extracts)
Urea/thiourea – for 2D gels
(small HSP)
RIPA (smooth muscle proteins)
Personalize lysis buffer
Peach et al, 2013. Methods in Mol Biol 869, 37-47.
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SDS to protein ratio
1.4 µg SDS per 1µg of protein
Recommended SDS to protein
ratio - 3:1
Protein concentration should be
assayed to prevent inadequate
protein to sample buffer ratios
Hames, B. D. (1990) in “Gel Electrophoresis of Proteins,”
Hames, B. D., and Rickwood, D., eds., pp. 1–147, Oxford
University Press
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Solubilization with
Laemmli buffer
SDS does not unfold some
proteases
Heat sample soon after adding LB
Avoids protein degradation by
proteases
1 pg of proteases degrades
proteins if not heated soon after
adding LB
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Heating with Laemmli
buffer
Do not heat sample at 100 ˚C > 5
min
Asp-Pro bond most susceptible for
cleavage by heat and acidic
conditions
75˚C for 5 min avoids D-P bond
cleavage and inactivates proteases Volkin et al, 1995. Methods Mol. Biol. 40, 35–63
Deutsch, 1976. Anal Biochem 71, 300–303
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Solubilizing
histones/membrane proteins
Heating with SDS LB alone may not
solubilize histones/memb proteins
6-8 M urea or Triton X-100
Insoluble material should be
removed by a 17,000 G spin (2 min)
This avoids streaking within gel
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Protein aggregates in SDS
samples
Reductant becomes partly
oxidized, upon cooling sample,
part of the cysteines unprotected
Back-folding and creation of
inter-polypeptide aggregates
Blurred zones, formation of
double bands
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Cooling sample to ~60 ˚C and
adding an alkylating agent
obviates this problem
Sharper bands
Artifacts abolished
Protein aggregates
(cont’d)
Galvani et al, Electrophoresis
2001, 22, 2066–2074
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Miscalculating cross-
linking factor
Pore size of a polyacrylamide gel (a)
total concentration of acrylamide T
(b) degree of cross-linking C
Mistakenly assume that given total
acrylamide concentration T is the
percentage of acrylamide per volume
And C (crosslinking factor) is the
percentage of bis per volume Westermeier R. Practical Proteomics, 2007;
Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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Miscalculating cross-
linking factor
a= mass of acrylamide in g
b= mass of bis in g
V= volume
Alternatively, use commercially available
acrylamide/bis stock solutions
Burgess, R.R. (2009) Methods Enzymol 463, 813-820. Review.
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Electrophoresis
Overloading and underloading
commonly encountered
Overloading - distorted bands in
lane, also in adjacent lanes
Underloading - poor detection of
minor bands
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Titrating running buffer in
SDS PAGE Use only Tris-base, glycine, and
SDS
Adjusting pH to 8.3 leads to
higher load of chloride
Longer protein separation times
Some zones remain poorly
resolved
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SDS-PAGE
Best to polymerize gel O/N at RT
Haeberle gel - greatly accelerated
rate of polyacrylamide cross-linking
Run gel in 5 min, Haeberle RB at 70 ˚C
Haeberle J R. BioTechniques 23, 638-640, 1997
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SDS-PAGE
Standard mini-gel, 2-4 hours
Pre-cast gel (4-20, 10%), in 10
min using heated Laemmli RB
Very recent results, showing
that electrophoresis, WB, and
immunoblotting - few hours
with actual run time of 60 min
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Transfer buffers
Towbin buffer system (25 mM Tris,
192 mM glycine, 20% methanol ,
none to 0.01% SDS)
CAPS buffer system (CAPS, 10 %
methanol pH 11) for transfer to
PVDF prior to in situ blot
sequencing
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Towbin transfer buffer
Transfer buffers without SDS are
better
Proteins can pass through
Immobilon-P membrane in the
presence of SDS
However, SDS needed (<0.01%)
during transfer of proteins that
precipitate
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Methanol in transfer
buffer
Methanol aids in stripping SDS from
proteins
It stabilizes the geometry of the gel
binding capacity of NC for
protein
Helps proteins to bind better to NC
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Methanol-less protein
transfer
Transferring high m. wt. proteins
Transferring conformation sensitive Ab
Enzyme activity needs to be
preserved
Drawback- PAGE gels swell in low-
ionic buffers
“bands” distorted if swelling occurs
during transfer
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Blotting from SDS-PAGE
Proteins eluted as anions
Membrane placed on anode
side of gel
Remove bubbles bet NC and
gel, prevent bald spots
Gel +filter+ pads tightly held
together
Good transfer, no band
distortion
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Do not exceed binding
capacity of membrane
Excess protein, weakly associated
with membrane, readily
accessible to antibody
Ensuing protein-antibody complex
wash off
Reduced signal
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Inefficient transfer of high
m. wt. proteins
Reversible gel cross-linkers, gel
depolymerization
Limited protease digestion during
transfer
Prolonged transfer with addition
of SDS (0.1%)
Heat mediated transfer
Gershoni and Palade, Anal Biochem 131:1-15, 1983
Kurien and Scofield, J Immunol Methods 266:127-33, 2002
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Preventing loss of low m.
wt. proteins
Low m. wt. proteins bind with low
affinities
Proteins lost during transfer or
washing
Cross-linking proteins to membrane
Use of 0.2 µm reduces “blow
through”
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Conclusions
Simple method
Potential of western blotting
greatly evolved
Huge number of ways to transfer
proteins
Has led to a deeper
understanding of protein-ligand
interaction
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Brought to you by the Science/AAAS Custom Publishing Office
Sponsored by:
Participating Experts
Pier Giorgio Righetti, Ph.D.
Polytechnic University of Milan
Milan, Italy
Biji T. Kurien, Ph.D.
University of Oklahoma Health
Sciences Center
Oklahoma City, OK
Webinar Series
Nick Thomas, Ph.D.
GE Healthcare
Cardiff, Wales
Western Blot Tips and Tricks
Filling the Gap Between Art and Science
July 30, 2014
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Imagination at work
Nick Thomas PhD Principal Scientist GE Healthcare Life Sciences
Western Blotting The Art & The Science
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Western Blotting Core technique in life science research
~37,000 labs worldwide ~60% of life sciences publications contain Western Blotting data Expression Characterisation Localisation Modification Diagnostics User intensive non-standardised workflow generates 35-45% variability in data Results depend on skill and experience with 25% failure rate
Pro
tein
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Western Blotting Need for improvement
Standardisation and improved reproducibility
Better quantitation
Systematised simplified workflow
Get it right each and every time
Free up research time
“Did I put the stack in the blotter the
wrong way round ?”
“Looks like I forgot the
blocking buffer again !”
“####! – Can I run it again
tonight?”
“Well I thought
that old wash buffer was OK”
“What the #### ?!?!?”
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Western Blotting From DAB to CCD
Started Western blotting with HRP colourimetric detection Applying photographic developing methods to Western blot processing First pre-stained molecular weight markers for SDS-PAGE and Western blotting Worked on development of Amersham™ ECL™ - the first commercially available chemiluminescence detection system
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You don't take a photograph, you make it. Ansel Adams 1902-1984
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Photographic Evolution Different science, same artistry
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Photographic Evolution Different science, same artistry
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Western Blotting Evolution 20th to 21st century technologies
• HRP & Alk Phos • Nitrocellulose • Low sensitivity • Non-quantitative • Low reproducibility
• Range of detection substrates for higher sensitivity
• PVDF membranes • X-ray film and CCD
imagers • Quantitation
possible
• Quantitative • Standardisation to
HKP or total protein loading
• Multiplexing for 2-3 proteins and/or post-translational modifications
Colourimetric Chemiluminescent Fluorescent
Film Digital Imaging
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Detection Improvements Dynamic range and multiplexing
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Fluorescence Multiplexing Protein phosphorylation
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Western Blotting Evolution Ready for the next stage
Integrate optimal technologies Systematise workflow Software integration for guiding, optimising and monitoring protocols to be; Standardised Quantitative Smart
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Western Blotting Evolution Systems Integration
Streamline Workflows
Remove errors
Better artistry Better science
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Western Blotting Evolution Different Science, Same Artistry
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GE, imagination at work, and GE monogram are trademarks of trademarks of General Electric Company or one of its subsidiaries. Amersham, Cy, CyDye, ECL, ECL Advance, ECL DualVue, ECL Prime, ECL Plex, ECL Select, Hybond, and Rainbow are trademarks of GE Healthcare companies. ECL Advance contains Lumigen TMA-6 substrate and is sold under license from Lumigen, Inc. ECL Plus contains Lumigen PS3 substrate and is sold under license from Lumigen, Inc. ECL Prime: This product or portions thereof is manufactured and sold under license from Cyanagen Srl and is subject of US patent 7,855,287, US Patent 7,803,573, and Italian application number TO2010A000580, together with other equivalent granted patents and patent applications in other countries. CyDye: This product is manufactured under an exclusive license from Carnegie Mellon University and is covered by US patent numbers 5,569,587 and 5,627,027. The purchase of CyDye products includes a limited license to use the CyDye products for internal research and development but not for any commercial purposes. A license to use the CyDye products for commercial purposes is subject to a separate license agreement with GE Healthcare. Commercial use shall include: 1. Sale, lease, license or other transfer of the material or any material derived or produced from it. 2. Sale, lease, license or other grant of rights to use this material or any material derived or produced from it. 3. Use of this material to perform services for a fee for third parties, including contract research and drug screening. If you require a commercial license to use this material and do not have one, return this material unopened to GE Healthcare Bio-Sciences AB, Bjorkgatan 30, SE-751 84 Uppsala, Sweden and any money paid for the material will be refunded. All goods and services are sold subject to the terms and conditions of sale of the company within GE Healthcare which supplies them. A copy of these terms and conditions is available on request. Contact your local GE Healthcare representative for the most current information.
©2008–2014 General Electric Company—All rights reserved. First published July 2014.
GE Healthcare UK Limited, Amersham Place, Little Chalfont, Buckinghamshire, HP7 9NA, UK
www.gelifesciences.com/artofwesternblotting
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To submit your
questions, type them
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Participating Experts
Sponsored by:
Brought to you by the Science/AAAS Custom Publishing Office
Webinar Series
Western Blot Tips and Tricks
Filling the Gap Between Art and Science
July 30, 2014
Pier Giorgio Righetti, Ph.D.
Polytechnic University of Milan
Milan, Italy
Biji T. Kurien, Ph.D.
University of Oklahoma Health
Sciences Center
Oklahoma City, OK
Nick Thomas, Ph.D.
GE Healthcare
Cardiff, Wales
![Page 67: Western Blot Tips and Tricks slides_Western...Western Blot Tips and Tricks Filling the Gap Between Art and Science Instructions for Viewers Webinar Series July 30, 2014 Brought to](https://reader031.fdocuments.us/reader031/viewer/2022021811/5c955b0d09d3f29c7b8c906f/html5/thumbnails/67.jpg)
For related information on this webinar topic, go to:
gelifesciences.com/artofwesternblotting
Look out for more webinars in the series at:
webinar.sciencemag.org
To provide feedback on this webinar, please e-mail your comments to [email protected]
Brought to you by the Science/AAAS Custom Publishing Office
Sponsored by:
Webinar Series
Western Blot Tips and Tricks
Filling the Gap Between Art and Science
July 30, 2014